RESUMO
Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.
Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Malária/tratamento farmacológico , Malária/parasitologia , Plasmodium/efeitos dos fármacos , Plasmodium/enzimologia , 1-Fosfatidilinositol 4-Quinase/química , 1-Fosfatidilinositol 4-Quinase/genética , 1-Fosfatidilinositol 4-Quinase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Citocinese/efeitos dos fármacos , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Ácidos Graxos/metabolismo , Feminino , Hepatócitos/parasitologia , Humanos , Imidazóis/metabolismo , Imidazóis/farmacologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Macaca mulatta , Masculino , Modelos Biológicos , Modelos Moleculares , Fosfatos de Fosfatidilinositol/metabolismo , Plasmodium/classificação , Plasmodium/crescimento & desenvolvimento , Pirazóis/metabolismo , Pirazóis/farmacologia , Quinoxalinas/metabolismo , Quinoxalinas/farmacologia , Reprodutibilidade dos Testes , Esquizontes/citologia , Esquizontes/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
A novel family of 1H-imidazol-2-yl-pyrimidine-4,6-diamines has been identified with potent activity against the erythrocyte-stage of Plasmodium falciparum (Pf), the most common causative agent of malaria. A systematic SAR study resulted in the identification of compound 40 which exhibits good potency against both wild-type and drug resistant parasites and exhibits good in vivo pharmacokinetic properties.
Assuntos
Antimaláricos/química , Plasmodium falciparum/efeitos dos fármacos , Pirimidinas/química , Animais , Antimaláricos/farmacocinética , Antimaláricos/farmacologia , Descoberta de Drogas , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Farnesoid X receptor (FXR) agonists are emerging as important potential therapeutics for the treatment of nonalcoholic steatohepatitis (NASH) patients, as they exert positive effects on multiple aspects of the disease. FXR agonists reduce lipid accumulation in the liver, hepatocellular inflammation, hepatic injury, and fibrosis. While there are currently no approved therapies for NASH, the bile acid-derived FXR agonist obeticholic acid (OCA; 6-ethyl chenodeoxycholic acid) has shown promise in clinical studies. Previously, we described the discovery of tropifexor (LJN452), the most potent non-bile acid FXR agonist currently in clinical investigation. Here, we report the discovery of a novel chemical series of non-bile acid FXR agonists based on a tricyclic dihydrochromenopyrazole core from which emerged nidufexor (LMB763), a compound with partial FXR agonistic activity in vitro and FXR-dependent gene modulation in vivo. Nidufexor has advanced to Phase 2 human clinical trials in patients with NASH and diabetic nephropathy.
Assuntos
Benzotiazóis/uso terapêutico , Ácido Quenodesoxicólico/análogos & derivados , Dieta Hiperlipídica/efeitos adversos , Isoxazóis/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Benzotiazóis/química , Ácido Quenodesoxicólico/química , Ácido Quenodesoxicólico/uso terapêutico , Cães , Humanos , Isoxazóis/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/etiologia , Estrutura Terciária de Proteína , Ratos , Resultado do TratamentoRESUMO
Screening our in-house compound collection using a cell based Plasmodium falciparum proliferation assay we discovered a known pan-kinase inhibitor scaffold as a hit. Further optimization of this series led us to a novel benzamide scaffold which was devoid of human kinase activity while retaining its antiplasmodial activity. The evolution of this compound series leading to optimized candidates with good cellular potency against multiple strains as well as decent in vivo profile is described in this Letter.
Assuntos
Antimaláricos/química , Benzamidas/química , Inibidores Enzimáticos/química , Fosfotransferases/antagonistas & inibidores , Plasmodium falciparum/enzimologia , Animais , Antimaláricos/síntese química , Antimaláricos/farmacologia , Benzamidas/síntese química , Benzamidas/farmacologia , Evolução Molecular Direcionada , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Plasmodium falciparum/efeitos dos fármacosRESUMO
Farnesoid X receptor (FXR) agonism is emerging as an important potential therapeutic mechanism of action for multiple chronic liver diseases. The bile acid-derived FXR agonist obeticholic acid (OCA) has shown promise in a phase 2 study in patients with nonalcoholic steatohepatitis (NASH). Here, we report efficacy of the novel nonbile acid FXR agonist tropifexor (LJN452) in two distinct preclinical models of NASH. The efficacy of tropifexor at <1 mg/kg doses was superior to that of OCA at 25 mg/kg in the liver in both NASH models. In a chemical and dietary model of NASH (Stelic animal model [STAM]), tropifexor reversed established fibrosis and reduced the nonalcoholic fatty liver disease activity score and hepatic triglycerides. In an insulin-resistant obese NASH model (amylin liver NASH model [AMLN]), tropifexor markedly reduced steatohepatitis, fibrosis, and profibrogenic gene expression. Transcriptome analysis of livers from AMLN mice revealed 461 differentially expressed genes following tropifexor treatment that included a combination of signatures associated with reduction of oxidative stress, fibrogenesis, and inflammation. Conclusion: Based on preclinical validation in animal models, tropifexor is a promising investigational therapy that is currently under phase 2 development for NASH.
RESUMO
Structure-based design was utilized to guide the early stage optimization of a substrate-like inhibitor to afford potent peptidomimetic inhibitors of the channel-activating protease prostasin. The first X-ray crystal structures of prostasin with small molecule inhibitors bound to the active site are also reported.
Assuntos
Serina Endopeptidases/efeitos dos fármacos , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/farmacologia , Técnicas de Química Combinatória , Cristalografia por Raios X , Mimetismo Molecular , Estrutura Molecular , Conformação Proteica , Inibidores de Serina Proteinase/química , Relação Estrutura-AtividadeRESUMO
The farnesoid X receptor (FXR) is a nuclear receptor that acts as a master regulator of bile acid metabolism and signaling. Activation of FXR inhibits bile acid synthesis and increases bile acid conjugation, transport, and excretion, thereby protecting the liver from the harmful effects of bile accumulation, leading to considerable interest in FXR as a therapeutic target for the treatment of cholestasis and nonalcoholic steatohepatitis. We identified a novel series of highly potent non-bile acid FXR agonists that introduce a bicyclic nortropine-substituted benzothiazole carboxylic acid moiety onto a trisubstituted isoxazole scaffold. Herein, we report the discovery of 1 (tropifexor, LJN452), a novel and highly potent agonist of FXR. Potent in vivo activity was demonstrated in rodent PD models by measuring the induction of FXR target genes in various tissues. Tropifexor has advanced into phase 2 human clinical trials in patients with NASH and PBC.
Assuntos
Benzotiazóis/farmacologia , Colestase/tratamento farmacológico , Isoxazóis/farmacologia , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/agonistas , Administração Oral , Animais , Benzotiazóis/uso terapêutico , Disponibilidade Biológica , Cães , Avaliação Pré-Clínica de Medicamentos/métodos , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Isoxazóis/uso terapêutico , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Piperidinas/química , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade , Triglicerídeos/sangueRESUMO
Our current understanding of the role and regulation of protease activity in normal and pathogenic processes is limited by our ability to measure and deconvolute their enzymatic activity. To address this limitation, an approach was developed that utilizes rhodamine-based fluorogenic substrates encoded with PNA tags. The PNA tags address each of the substrates to a predefined location on an oligonucleotide microarray through hybridization, thus allowing the deconvolution of multiple signals from a solution. A library of 192 protease substrates was prepared by split and mix combinatorial synthesis. The methodology and validation of this approach for profiling proteolytic activity from single proteases and from those in crude cell lysates as well as clinical blood samples is described.
Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Ácidos Nucleicos Peptídicos/genética , Humanos , Hidrólise , Células Jurkat , Peptídeo Hidrolases/química , Biblioteca de Peptídeos , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/metabolismo , Proteômica/métodos , Especificidade por Substrato/genéticaRESUMO
Piezo ion channels are activated by various types of mechanical stimuli and function as biological pressure sensors in both vertebrates and invertebrates. To date, mechanical stimuli are the only means to activate Piezo ion channels and whether other modes of activation exist is not known. In this study, we screened ~3.25 million compounds using a cell-based fluorescence assay and identified a synthetic small molecule we termed Yoda1 that acts as an agonist for both human and mouse Piezo1. Functional studies in cells revealed that Yoda1 affects the sensitivity and the inactivation kinetics of mechanically induced responses. Characterization of Yoda1 in artificial droplet lipid bilayers showed that Yoda1 activates purified Piezo1 channels in the absence of other cellular components. Our studies demonstrate that Piezo1 is amenable to chemical activation and raise the possibility that endogenous Piezo1 agonists might exist. Yoda1 will serve as a key tool compound to study Piezo1 regulation and function.
Assuntos
Canais Iônicos/agonistas , Canais Iônicos/metabolismo , Mecanotransdução Celular/fisiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Fluorescência , Ensaios de Triagem em Larga Escala , Humanos , CamundongosRESUMO
A high throughput screening campaign identified 5-(2-chlorophenyl)indazole compound 4 as an antagonist of the transient receptor potential A1 (TRPA1) ion channel with IC50 = 1.23 µM. Hit to lead medicinal chemistry optimization established the SAR around the indazole ring system, demonstrating that a trifluoromethyl group at the 2-position of the phenyl ring in combination with various substituents at the 6-position of the indazole ring greatly contributed to improvements in vitro activity. Further lead optimization resulted in the identification of compound 31, a potent and selective antagonist of TRPA1 in vitro (IC50 = 0.015 µM), which has moderate oral bioavailability in rodents and demonstrates robust activity in vivo in several rodent models of inflammatory pain.
Assuntos
Indazóis/química , Proteínas do Tecido Nervoso/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Administração Oral , Analgésicos/química , Analgésicos/farmacocinética , Analgésicos/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Disponibilidade Biológica , Células CHO , Canais de Cálcio , Cricetulus , Adjuvante de Freund , Humanos , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Indazóis/farmacocinética , Indazóis/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Mostardeira , Óleos de Plantas , Ratos Wistar , Especificidade da Espécie , Relação Estrutura-Atividade , Canal de Cátion TRPA1 , Canais de Cátion TRPC/antagonistas & inibidoresRESUMO
Imidazopyridine 1 was identified from a phenotypic screen against P. falciparum (Pf) blood stages and subsequently optimized for activity on liver-stage schizonts of the rodent parasite P. yoelii (Py) as well as hypnozoites of the simian parasite P. cynomolgi (Pc). We applied these various assays to the cell-based lead optimization of the imidazopyrazines, exemplified by 3 (KAI407), and show that optimized compounds within the series with improved pharmacokinetic properties achieve causal prophylactic activity in vivo and may have the potential to target the dormant stages of P. vivax malaria.
RESUMO
On the basis of the initial success of optimization of a novel series of imidazolopiperazines, a second generation of compounds involving changes in the core piperazine ring was synthesized to improve antimalarial properties. These changes were carried out to further improve the potency and metabolic stability of the compounds by leveraging the outcome of a set of in vitro metabolic identification studies. The optimized 8,8-dimethyl imidazolopiperazine analogues exhibited improved potency, in vitro metabolic stability profile and, as a result, enhanced oral exposure in vivo in mice. The optimized compounds were found to be more efficacious than the current antimalarials in a malaria mouse model. They exhibit moderate oral exposure in rat pharmacokinetic studies to achieve sufficient multiples of the oral exposure at the efficacious dose in toxicology studies.
Assuntos
Antimaláricos/farmacologia , Imidazóis/farmacologia , Malária Falciparum/tratamento farmacológico , Piperazinas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/síntese química , Antimaláricos/química , Antimaláricos/farmacocinética , Disponibilidade Biológica , Células CACO-2 , Humanos , Imidazóis/síntese química , Imidazóis/química , Imidazóis/farmacocinética , Malária Falciparum/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Piperazinas/síntese química , Piperazinas/química , Piperazinas/farmacocinética , Plasmodium falciparum/metabolismo , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
Starting from a hit series from a GNF compound library collection and based on a cell-based proliferation assay of Plasmodium falciparum, a novel imidazolopiperazine scaffold was optimized. SAR for this series of compounds is discussed, focusing on optimization of cellular potency against wild-type and drug resistant parasites and improvement of physiochemical and pharmacokinetic properties. The lead compounds in this series showed good potencies in vitro and decent oral exposure levels in vivo. In a Plasmodium berghei mouse infection model, one lead compound lowered the parasitemia level by 99.4% after administration of 100 mg/kg single oral dose and prolonged mice survival by an average of 17.0 days. The lead compounds were also well-tolerated in the preliminary in vitro toxicity studies and represents an interesting lead for drug development.
Assuntos
Antimaláricos/síntese química , Imidazóis/síntese química , Piperazinas/síntese química , Aminoácidos/síntese química , Aminoácidos/química , Aminoácidos/farmacologia , Compostos de Anilina/síntese química , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Derivados de Benzeno/síntese química , Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , Linhagem Celular , Resistência a Medicamentos , Feminino , Humanos , Imidazóis/química , Imidazóis/farmacologia , Concentração Inibidora 50 , Malária/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Piperazinas/química , Piperazinas/farmacologia , Plasmodium berghei , Plasmodium falciparum/efeitos dos fármacos , Ratos , Relação Estrutura-AtividadeRESUMO
Prostasin or human channel-activating protease 1 has been reported to play a critical role in the regulation of extracellular sodium ion transport via its activation of the epithelial cell sodium channel. Here, the structure of the extracellular portion of the membrane associated serine protease has been solved to high resolution in complex with a nonselective d-FFR chloromethyl ketone inhibitor, in an apo form, in a form where the apo crystal has been soaked with the covalent inhibitor camostat and in complex with the protein inhibitor aprotinin. It was also crystallized in the presence of the divalent cation Ca(+2). Comparison of the structures with each other and with other members of the trypsin-like serine protease family reveals unique structural features of prostasin and a large degree of conformational variation within specificity determining loops. Of particular interest is the S1 subsite loop which opens and closes in response to basic residues or divalent ions, directly binding Ca(+2) cations. This induced fit active site provides a new possible mode of regulation of trypsin-like proteases adapted in particular to extracellular regions with variable ionic concentrations such as the outer membrane layer of the epithelial cell.
Assuntos
Domínio Catalítico , Cátions Bivalentes/metabolismo , Serina Endopeptidases/química , Aprotinina/metabolismo , Cálcio/metabolismo , Quimotripsina/metabolismo , Cristalografia por Raios X , Ésteres , Gabexato/análogos & derivados , Gabexato/metabolismo , Guanidinas , Humanos , Inibidores de Proteases/metabolismo , Conformação Proteica , Alinhamento de Sequência , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Especificidade por SubstratoRESUMO
Peptidic, non-covalent inhibitors of lysosomal cysteine protease cathepsin S (1 and 2) were investigated due to low oral bioavailability, leading to an improved series of peptidomimetic inhibitors. Utilizing phenyl succinamides as the P2 residue increased the oral exposure of this lead series of compounds, while retaining selective inhibition of the cathepsin S isoform. Concurrent investigation of the P1 and P2 subsites resulted in the discovery of several potent and selective inhibitors of cathepsin S with good pharmacokinetic properties due to the elimination of saturated aliphatic P2 residues.
Assuntos
Amidas/síntese química , Catepsinas/antagonistas & inibidores , Inibidores de Proteases/síntese química , Amidas/química , Amidas/farmacocinética , Amidas/farmacologia , Animais , Desenho de Fármacos , Masculino , Estrutura Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/farmacologia , Ratos , Ratos Wistar , Relação Estrutura-Atividade , SuccinatosRESUMO
We report a novel series of noncovalent inhibitors of cathepsin S. The synthesis of the peptidomimetic scaffold is described and structure-activity relationships of P3, P1, and P1' subunits are discussed. Lead optimization to a non-peptidic scaffold has resulted in a new class of potent, highly selective, and orally bioavailable cathepsin S inhibitors.
Assuntos
Carbamatos/síntese química , Carbamatos/farmacologia , Catepsinas/antagonistas & inibidores , Oligopeptídeos/síntese química , Inibidores de Proteases/síntese química , Administração Oral , Animais , Disponibilidade Biológica , Carbamatos/farmacocinética , Humanos , Masculino , Mimetismo Molecular , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/farmacologia , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
A systematic study of anilines led to the discovery of a metabolically robust fluoroindoline replacement for the alkoxy aniline toxicophore in 1. Investigations of the P1 pocket resulted in the discovery of a wide tolerance of functionality leading to the discovery of 11 as a potent and selective inhibitor of cathepsin S.
Assuntos
Amidas/farmacologia , Catepsinas/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Desenho de Fármacos , Amidas/síntese química , Amidas/química , Aminação , Derivados de Benzeno/síntese química , Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , Sítios de Ligação , Catepsina K , Catepsina L , Catepsinas/química , Catepsinas/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Etanol/química , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The synthesis and structure-activity relationship of a series of arylaminoethyl amide cathepsin S inhibitors are reported. Optimization of P3 and P2 groups to improve overall physicochemical properties resulted in significant improvements in oral bioavailability over early lead compounds. An X-ray structure of compound 37 bound to the active site of cathepsin S is also reported.
Assuntos
Amidas/síntese química , Amidas/farmacologia , Catepsinas/antagonistas & inibidores , Administração Oral , Amidas/farmacocinética , Animais , Sítios de Ligação , Disponibilidade Biológica , Cristalografia por Raios X , Éteres Cíclicos/síntese química , Éteres Cíclicos/farmacologia , Humanos , Masculino , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/farmacologia , Ratos , Ratos Wistar , Relação Estrutura-Atividade , ZincoRESUMO
A series of N(alpha)-2-benzoxazolyl-alpha-amino acid-(arylaminoethyl)amides were identified as potent, selective, and noncovalent inhibitors of cathepsin S. Structure-activity relationships including strategies for modulating the selectivities among cathepsins S, K, and L, and in vivo pharmacokinetics are discussed. A X-ray structure of compound 3 bound to the active site of cathepsin S is also reported.
Assuntos
Amidas/farmacologia , Catepsinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos/farmacologia , Amidas/química , Animais , Catepsinas/química , Catepsinas/genética , Catepsinas/fisiologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Compostos Heterocíclicos/química , Camundongos , Camundongos Knockout , Modelos Moleculares , RatosRESUMO
Regulated proteolysis by the two-component NS2B/NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1' and P3'. In contrast, the P2' and P4' substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.